ABSTRACT The global burden of hepatitis C virus (HCV) infection is at 71 million with an annual rate of 1.75 million new infections each year. In the US, HCV infection is increasing in young adults because of injection drug use. A preventive vaccine is needed in spite of major advances in the development of direct acting antivirals (DAAs) for the treatment of HCV infections. The HCV envelope glycoproteins E1 and E2 form a heterodimer and higher- order assemblies on the native virus. This complex is the key antigen in candidate HCV vaccines, and comprises the target of the antibody response to HCV, yet strikingly little is known about the details of its assembly and structure. Detailed information on HCV glycoprotein E1 and E2 assembly determinants would greatly advance our knowledge of HCV structure and the anti-HCV immune response, and would enable rational vaccine design to engineer stable E1E2 assembly and epitope presentation. Various studies have demonstrated that the C- terminal transmembrane domains of E1 and E2 are critical for E1E2 complex assembly, yet the presence of the transmembrane domains hinders detailed structural studies, biochemical characterization, and vaccine development. We propose to design soluble E1E2 assemblies containing functional replacements for the transmembrane domains which promote native oligomerization, analogous to successful efforts to stabilize other transmembrane viral glycoproteins such as influenza hemagglutinin and RSV F. We propose an iterative, interdisciplinary approach to design soluble native E1E2 assemblies. In Aim 1, scaffolds will be selected from known oligomeric structures and defined architectures. Computational modeling and design methods will be used both to optimize scaffolds and to generate novel scaffolds based on initial experimental data. In Aim 2, purified soluble E1E2 complexes using the best scaffolds from Aim 1 will be produced using mammalian cell expression systems. Native E1E2 assembly will be confirmed using binding assays to a panel of antibodies targeting conformational epitopes on E1, E2 and E1E2, as well as HCV coreceptors. Biophysical assays will be used to assess size, oligomerization, and other properties of designed constructs. Those with native-like antigenicity will be structurally characterized by cryo-EM, and an in vivo immunogenicity study will be used to confirm that top soluble E1E2 designs elicit robust neutralizing antibodies, and to determine correlates between structure, antigenicity, biophysical properties, and immunogenicity. Providing a proof of concept, one of our initial designs exhibits reactivity to multiple human antibodies targeting conformational epitopes on the native E1E2 complex. This work will establish a platform for E1E2 rational design and structural biology. These studies will thereby enable the study of E1E2 complex assembly, recognition by the immune system, and role in viral entry across the diverse genetic landscape of HCV. Moreover, the principles learned through this work will provide the framework for further efforts aimed at rational design of an E1E2 glycoprotein HCV vaccine.